We have carried out a systematic study on the guanidinium chloride- and urea-induced unfolding of glucose oxidase from Aspergillus niger, an acidic dimeric enzyme, using various optical spectroscopic techniques, enzymatic activity measurements, glutaraldehyde cross-linking, and differential scanning calorimetry. The urea-induced unfolding of GOD was a two-state process with dissociation and unfolding of the native dimeric enzyme molecule occurring in a single step. On the contrary, the GdmCl-induced unfolding of GOD was a multiphasic process with stabilization of a conformation more compact than the native enzyme at low GdmCl concentrations and dissociation along with unfolding of enzyme at higher concentrations of GdmCl. The GdmCl-stabilized compact dimeric intermediate of GOD showed an enhanced stability against thermal and urea denaturation as compared to the native GOD dimer. Comparative studies on GOD using GdmCl and NaCl demonstrated that binding of the Gdm(+) cation to the enzyme results in stabilization of the compact dimeric intermediate of the enzyme at low GdmCl concentrations. An interesting observation was that a slight difference in the concentration of urea and GdmCl associated with the unfolding of GOD was observed, which is in violation of the 2-fold rule for urea and GdmCl denaturation of proteins. This is the first report where violation of the 2-fold rule has been observed for a multimeric protein. Study holds ProTherm entries: 13010, 13011 Extra Details: optical spectroscopic technique; two-state process; dimeric intermediate;,multiphasic process
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:44 p.m.
|Number of data points||2|
|Proteins||Glucose oxidase ; Glucose oxidase|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: Cm|
|Libraries||Mutations for sequence SNGIEASLLTDPKDVSGRTVDYIIAGGGLTGLTTAARLTENPNISVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDNVAAYSLQAERARAPNAKQIAAGHYFNASCHGVNGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDFGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNGTTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTATVRSRITSAGAGQGQAAWFATFNETFGDYSEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVNHNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLHHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLSAWTEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKISDAILEDYASMQ|